Biomedical Engineering Reference
In-Depth Information
0
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3.14
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0.0
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3.14
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0
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2
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FIGuRE 15.25
Amplitude.and.phase.of.the.wavefront.in.the.back.pupil.plane.calculated.using.phase.retrieval.
from.a.200.nm.luorescent.bead.injected.40.μm.below.the.surface.of.a.
Drosophila
.embryo..(a).Image.of.the.bead.used.
in.phase.retrieval..(i).A.lateral.cross.section,.and.(ii).a.longitudinal.cross.section..(b).Phase.(radians).and.amplitude.
(normalized).of.the.wavefront.in.the.back.pupil.plane..(c).Point.spread.functions.calculated.from.the.wavefront.in.
the.back.pupil.plane..he.top.image.is.a.lateral.in-focus.cross.section,.and.the.bottom.image.is.a.longitudinal.cross.
section..(i).PSF.from.the.wavefront.in.(b)..(ii).PSF.function.from.the.wavefront.in.(b).with.the.phase.corrected.(set.
to.0)..(iii).PSF.from.wavefront.with.uniform.amplitude.and.lat.phase.
of.the.wavefront.is.still.distorted,.and.amplitude.distortions.cannot.be.corrected.by.DMs..If.both.the.
amplitude.and.phase.could.be.corrected.(keeping.the.integrated.intensity.the.same),.the.peak.intensity.
would.increase.by.a.factor.of.6.8.(Figure.15.25c(iii))..(Amplitude.correction.could.be.made.by.attenuat-
ing.the.strong.pupil.regions.to.equal.the.weaker.regions,.obviously.reducing.the.integrated.intensity.).As.
can.be.seen,.the.image.can.be.signiicantly.improved.by.only.correcting.the.phase.
15.3.6 Phase Diversity
An.important.drawback.of.wavefront.sensing.using.phase.retrieval.is.that.a.point-like.source.is.required.
to.determine.the.wavefront..Because.there.is.a.lack.of.point.sources.in.biological.samples,..determining.
the.wavefront.from.extended.images.is.a.signiicant.advantage..Phase.diversity.is.a.technique.that.does.
not. require. a. coherent. source. or. PSF. (Gonsalves. 1982;. Paxman. et. al.. 1992),. and. thus. lends. itself. to.
.wavefront.estimation.and.correction.when.imaging.extended.objects..Phase.diversity.has.been.used.in.
used.in.astronomy.(Paxman.et.al..1996),.but.has.not.yet.been.used.extensively.in.microscopy.
In.phase.diversity,.two.or.more.images.are.taken,.each.with.a.known.phase.aberration.introduced..
he. aberrations. and. the. object. are. estimated. by. inding. the. combination. of. object. and. PSF. that. best.
reproduces.all.the.measured.data..In.general,.this.requires.searching.the.entire.parameter.space.of.both.
the.image.and.the.PSF,.but.for.the.case.of.images.with.Gaussian.noise,.the.image.degrees.of.freedom.can.
be.eliminated.and.the.cost.function.can.be.minimized.by.searching.over.only.the.aberration.parameters.
(Paxman.et.al..1992).
